Project Summary This project is designed to reveal how the cancer-driver mutations found in the genes encoding central metabolic enzymes cause epigenetic consequences in the multiple types of cancers in which they are found. Work in the field indicates that mutations affecting one of these enzymes, isocitrate dehydrogenase, produce an onco-metabolite known a 2-hydroxyglutarate (2-HG) which can inhibit enzymes that remove methyl modifications of DNA and histones. Which methylated targets play critical roles in cancers is unclear. The PI has show that the very same mutations found in human cancer, when engineered into the yeast Saccharomyces cerevisiae produce 2-HG. He has also found a yeast gene that encodes an enzyme that metabolizes 2- HG. Using an ingenious assay for pertubations of heterochromatin, the overproduction caused by these mutations appears to hyper-stabilize heterochromatin, not by inhibiting DNA demethylation, but by inhibiting demethylation of a specific methylated species of lysine 36 on histone H3. The proposed experiments extend these genetic inferences by direct biochemical tests. They will test whether 2-HG is affecting any of the other structures of chromatin by well- established genomic methods that they are expert in. Further aims extend their surprising discovery that 2-HG is also made by wild-type cells. By harnessing the power of systematic genetic array (SGA) technology, they will identify the natural role of 2-HG in cells by identifying all genes that, when mutant, render cells sensitive to elevated 2-HG. Yeast models for the cancer-driving alleles of succinate dehydrogenase and fumarase will be made and tested for epigenetic impacts, and if found, the mechanism of the impact will be established. Building upon success with the yeast model of isocitrate dehydrogenase mutants, the PI has completed a successful pilot screen that identified 5 genes so far that affect heterochromatin stability through effects on metabolism, and propose completing the screen with an estimated recovery or 50- 100 genes, representing a near-comprehensive set of intersections betweeen metabolism and epigenetics. The mechanism behind each perturbations's epigenetic effect will be determined.